Malassezia colonisation on a reconstructed human epidermis: Imaging studies.

BACKGROUND: Biofilm formation represents a major microbial virulence attribute especially at epithelial surfaces such as the skin. Malassezia biofilm formation at the skin surface has not yet been addressed. OBJECTIVE: The present study aimed to evaluate Malassezia colonisation pattern on a reconstructed human epidermis (RhE) by imaging techniques. METHODS: Malassezia clinical isolates were previously isolated from volunteers with pityriasis versicolor and seborrhoeic dermatitis. Yeast of two strains of M furfur and M sympodialis were inoculated onto the SkinEthic™ RHE. The tissues were processed for light microscopy, wide-field fluorescence microscopy and scanning electron microscopy. RESULTS: Colonisation of the RhE surface with aggregates of Malassezia yeast entrapped in a multilayer sheet with variable amount of extracellular matrix was unveiled by imaging techniques following 24, 48, 72 and 96 hours of incubation. Whenever yeast were suspended in RPMI medium supplemented with lipids, the biofilm substantially increased with a dense extracellular matrix in which the yeast cells were embedded. Slight differences were found in the biofilm architectural structure between the two tested species with an apparently higher entrapment and viscosity in M furfur biofilm. CONCLUSION: Skin isolates of M furfur and M sympodialis were capable of forming biofilm in vitro at the epidermal surface simulating in vivo conditions. Following 24 hours of incubation, without added lipids, rudimental matrix was barely visible, conversely to the reported at plastic surfaces. The amount of biofilm apparently increased progressively from 48 to 96 hours. A structural heterogeneity of biofilm between species was found.

The evaluation of local oxidative/nitrosative stress in patients with pityriasis versicolor: a preliminary study.

Yeast of Malassezia, members of the microbiologic flora of the skin, cause pityriasis versicolor and have also been implicated in the pathogenesis of other superficial dermatoses. The mechanisms by which the yeast cause these dermatoses, however, are not yet clear, and there have been no studies on the interaction between fungi and keratinocytes, especially the effects of fungi on the production of reactive oxygen/nitrogen species by human keratinocytes. This is the first study we aimed to evaluate the role of oxidative/nitrosative stress in affected skin areas in patients with pityriasis versicolor. Sixty pityriasis versicolor patients and 60 healthy subjects as control group were enrolled. Scraping samples were obtained from the lesional skin areas in patients and from the normal skin areas in controls. The distribution of lesions of pityriasis versicolor was mild in all patients. We have isolated two Malassezia species: M. furfur being isolated in 75% of patients, followed by M. globosa (25%). The levels of oxidative/nitrosative stress biomarkers were significantly higher in patients than the controls (P < 0.001). Our results demonstrated that pityriasis versicolor is associated with significant alteration in oxidant/antioxidant balance. It might be secondary consequence of pityriasis versicolor infection or contributing factor in its pathogenesis.

The spectrum of Malassezia species isolated from students with pityriasis vesicolor in Nigeria.

Pityriasis versicolor (PV) is a common superficial fungal infection of the skin caused by Malassezia. Initially M. furfur was suggested as its main aetiological agent; however, more recent studies suggest M. globosa as the dominant species. The possibility of a variance in predominant species based on geographical basis has not been fully evaluated. The objective of this study was to identify the Malassezia species on affected and non-affected skin of students with PV who reside in a tropical environment (Abuja, Nigeria) and correlate them to clinical characteristics. In this study, the literature on prevalence of Malassezia genus in PV was also reviewed. Samples were taken from 304 PV lesions and 110 normal appearing skin. Microscopy, culture and identification of Malassezia species utilising polymerase chain reaction-restriction fragment length polymorphism analysis were performed. Three Malassezia species were detected in PV with the major species being M. furfur. On normal appearing skin, M. furfur (77.6%) and M. restricta (10.4%) were both detected. No case of M. globosa was identified in this study. There was no significant difference between species identified and clinical features of PV. M. furfur is probably still the most predominant species causing PV in the tropical environment.

[Cutaneous Malassezia infections and Malassezia associated dermatoses: An update]. / Kutane Malassezia-Infektionen und Malassezia-assoziierte Dermatosen : Ein Update.

The lipophilic yeast fungus Malassezia (M.) spp. is the only fungal genus or species which is part of the physiological human microbiome. Today, at least 14 different Malassezia species are known; most of them can only be identified using molecular biological techniques. As a facultative pathogenic microorganism, Malassezia represents the causative agent both of superficial cutaneous infections and of blood stream infections. Pityriasis versicolor is the probably most frequent infection caused by Malassezia. Less common, Malassezia folliculitis occurs. There is only an episodic report on Malassezia-induced onychomycosis. Seborrhoeic dermatitis represents a Malassezia-associated inflammatory dermatosis. In addition, Malassezia allergenes should be considered as the trigger of "Head-Neck"-type atopic dermatitis. Ketoconazole possesses the strongest in vitro activity against Malassezia, and represents the treatment of choice for topical therapy of pityriasis versicolor. Alternatives include other azole antifungals but also the allylamine terbinafine and the hydroxypyridone antifungal agent ciclopirox olamine. "Antiseborrhoeic" agents, e.g. zinc pyrithione, selenium disulfide, and salicylic acid, are also effective in pityriasis versicolor. The drug of choice for oral treatment of pityriasis versicolor is itraconazole; an effective alternative represents fluconazole. Seborrhoeic dermatitis is best treated with topical medication, including topical corticosteroids and antifungal agents like ketoconazole or sertaconazole. Calcineurin inhibitors, e.g. pimecrolimus and tacrolimus, are reliable in seborrhoeic dermatitis, however are used off-label.

[The use of calcofluor white for studying Malassezia species by direct microscopy]. / Empleo de blanco de calcoflúor para el estudio de las especies de Malassezia por microscopía directa.

Fungi of Malassezia genus are known as normal flora in human beings. However, different pathologies due to Malassezia, have been described. Traditionally, early diagnosis was delayed because of the difficulties in culture isolation of these organisms. The aim of this work, is to evaluate the technique of observation microscopy with calcofluor, for identification of Malassezia in both, clinical samples and isolates. In comparison to traditional method of direct examination with lactophenol-blue, calcofluor method offers an advantage because it turns easier the observation of fungal elements and its budding pattern. This technique contributes then, to identify species of Malassezia. The analysis of clinical specimens with calcofluor followed by observation under fluorescence microscopy is a simple and rapid method for the identification, and contribute therefore to the early diagnosis.

[Malassezia. Current knowledge and study perspectives]. / Malassezia. Estado del conocimiento y perspectivas en su estudio.

The study of Malassezia has been postponed for many years due to its nutritional requirements and its morphological variability. Molecular biology methods led to a taxonomic revision of this genus and to a new interest for its clinical importance. Nowadays, 11 Malassezia species are known, 10 are lipophilic, having similar morphological, physiological and biochemical characteristics, therefore, conventional techniques are not useful to differentiating them. Molecular methods are an accurate tool in the identification and they lead to a better knowledge of the ecology and epidemiology of this genus. Noteworthy antifungal susceptibility variations have been observed in some species, although there is not a standard method for these yeasts. There are few data about their biochemical characteristics, and the enzymes they produce might be important virulence factors, favouring host tissue invasion. Malassezia has been recognised as a member of the normal human and animal skin. Its implication in pathologic processes, including skin diseases to systemic infections, is the main issue in current investigations in order to determine the real pathogenic role of these yeasts.

Is transmission electron microscopy (TEM) a promising approach for qualitative and quantitative investigations of polymyxin B and miconazole interactions with cellular and subcellular structures of Staphylococcus pseudintermedius, Escherichia coli, Pseudomonas aeruginosa and Malassezia pachydermatis?

Antimicrobial therapy using a combination of polymyxin B and miconazole is effective against the main bacterial pathogens associated with otitis externa in dogs, and a synergistic effect of both drugs has been shown previously. The objective of the present investigation was to visualize ultrastructural changes after exposure of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus pseudintermedius and Malassezia pachydermatis to polymyxin B and miconazole by transmission electron microscopic (TEM). For this, cultures of E. coli, P. aeruginosa, S. pseudintermedius and M. pachydermatis were exposed to polymyxin B and miconazole, alone or in combination for 24 h. Ultrastructural changes were observed most frequently in the cell envelope of the four microorganisms. Exposure to polymyxin B seemed to cause more damage than miconazole within the range of concentrations applied. Treatment resulted in changes of the cell size: in E. coli, cell size increased significantly after treatment with either compound alone; in P. aeruginosa, cell size decreased significantly after treatment with polymyxin B and with miconazole; exposure of S. pseudintermedius to miconazole caused a decrease in cell size; in M. pachydermatis, cell size increased significantly after treatment with polymyxin B.; in E.coli, S. pseudintermedius and M. pachydermatis, cell size changed highly significant, in P. aeruginosa significantly after exposure to the combination of both compounds. In conclusion, by using a different approach than previous investigations, this study confirmed a clear combinatory effect of polymyxin B and miconazole against the tested microorganisms involved in canine otitis externa. It is the first time that visualization technologies were applied to compare the effect of single drugs to their combinatory effects on cellular and subcellular entities of selected bacterial and yeast species.

Dimorphism of Pityrosporum orbiculare in a defined culture medium.

Dimorphism of Pityrosporum orbiculare was induced in an artificial culture medium which consisted of 0.05 M glycine in 0.03-0.06 M ammonium phosphate buffer (pH 5.6), salts, glucose, and Tween-80. Of the strains tested, 78 to 85% produced hyphal elements from up to 8.5% of yeast cells. Morphologic features of P. orbiculare in culture were examined by scanning electron microscopy and compared with the appearance of fungus from clinical lesions of tinea versicolor. Morphologically, the fungi obtained in vivo and in vitro were identical.

Induction of hyphae in cultures of Pityrosporum by cholesterol and cholesterol esters.

The effect of cholesterol and cholesterol esters on Pityrosporum cultures has been studied. A mixture of 0.25% to 2.0% of cholesterol:cholesteryl stearate:glyceryl monostearate (2.0:1.5:2.0) added to Bacto Yeast Morphology Agar plus oleic acid was able to induce hyphae in cultures of both Pityrosporum orbiculare and P. ovale. This result is discussed with respect to the cholesterol effect on cell membranes and to the occurrence of cholesterol and cholesterol esters in the scaling patches of Pityriasis versicolor.

Pityrosporum isolate from the upper respiratory tract.

A 49-year-old white man experienced maxillary sinus osteitis after removal of a portion of the maxilla containing root tips from a previous molar tooth extraction. After the patient had received long-term antibiotic therapy for that infection, a yeast-like organism was observed on smears from the patient's left nasal passage and its surgically enlarged communication with the maxillary sinus. The fungus has appeared on all subsequent smears, but early attempts to culture the organism were unsuccessful. A lipophilic yeast-like fungus, morphologically similar to the organisms seen on the direct smears and consistent with the genus Pityrosporum, was isolated in culture at this facility. Electron microscopy of the organism revealed cellular morphologic features believed to be unique to the Pityrosporum genus, thereby confirming the identity of the isolate as a member of that genus.

Scanning electron microscopic evaluation of tinea versicolor. Effects of treatment with miconazole nitrate and clotrimazole.

A study of tinea versicolor organisms was conducted in one patient by scanning electron microscopy. Morphologic changes in the organisms during treatment with topical clotrimazole and miconazole nitrate consisted of initial wrinkling of the surface, followed by fragmentation. These changes may have reflected cell wall alterations resulting from inhibited ergosterol synthesis induced by both treatment agents. The two drugs were effective in eliminating the organisms.

Scanning electron microscopy of Malassezia furfur in tinea versicolor.

Morphologic features of Malassezia(M.) furfur in the horny layer from clinical lesions of tinea versicolor were examined by scanning electron microscopy and compared with the appearance of fungus in the horny layer from normal skin and in culture. In skin lesions of tinea versicolor, M. furfur showed a variety of growth and reproduction patterns. Although the main patterns were budding yeast forms, various patterns suggesting yeast-mycelial conversion were observed and mycelial hyphae were more prominent in the deeper horny layer than in the superficial layers. However, in the skin of normal persons and in culture, M. furfur existed only as yeast forms and no mycelial hyphae or yeast-mycelial conversion forms were seen. This suggests that the morphologic change of M. furfur, from a yeast form to a mycelial hypha one, may play a role in the induction of the clinical lesion of tinea versicolor.

Malassezia pachydermatis isolated from a South American sea lion (Otaria byronia) with dermatitis.

A fungus was isolated from the skin of an Otaria byronia and from the water of the pool in which the animal was kept. It formed creamy colonies with soft texture on Dixon agar and grew well without supplements of long-chain fatty acids. Cells were ovoid to cylindrical in shape, budded from a broad base, and budded and divided at the same location. Thus, the isolate was identified as M. pachydermatis. We compared this very rare isolate from a marine mammal with four strains of M. pachydermatis using the freeze-etching electron-microscopy technique. The cells showed the same characteristic ring-swellings on the protoplasmic membrane on the neck site between the mother and the daughter parts, and the same accumulation of circumvallate bulgings in a small area near the straight sections of spiral grooves as four reference strains. Thus, in terms of morphology and ultrastructure, the isolate could be regarded as a typical M. pachydermatis.

Attachment of Malassezia pachydermatis to the ear dermal cells in canine otitis externa.

To investigate the predominance of Malassezia pachydermatis (M. pachydermatis) as a causative agent of canine otitis externa, ear cerumen samples were observed for adhesion of M. pachydermatis to the cornified epithelial cells by light and electron microscopes. The yeasts appeared not to adhere to the cornified epithelial cells directly, but they seemed to exist in the proximity of the epithelial cells with an electron opaque halo-like space around them. Polysaccharide and lipid staining techniques were conducted to identify the substances existing in that space. Lipid substances, not saccharides, were observed around the yeasts and the cornified epithelial cells. These results suggested that in the canine ear canal malassezia yeast attachment to the cornified epithelial cells is mediated by lipids.

[Ultrastructure of fungal plasma membranes as revealed by freeze-replica].

This is a short review of fungal plasma membrane ultrastructure as revealed by freeze-replica. Characteristic features of growth polarity, growth phases, cell division, and ultrastructural characteristics after antibiotic treatments have been described.